RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
A growing list of naturally leaderless mRNAs has been reported to occur in all three domains of life. These leaderless mRNAs lack a 5' untranslated region (UTR) and are unable to establish the SD-ASD base-pairing between mRNA and 16S rRNA. Nevertheless, some of these leaderless mRNAs are efficiently translated. In addition, some mRNAs have very short untranslated regions (sUTR, length = 1-4 nt) upstream to the start codon. Leaderless cI-lacZ reporter constructs with added sUTRs were used to study the effect of sUTRs on expression. We observed a decrease in expression upon addition of sUTRs, with less than 5% expression when the sUTR length was 6nts. Further investigation revealed a direct correlation between decreased expression and decreased ribosome binding to the 5’-recessed AUG start codon. Additional investigation focused on the influence of the nucleotide composition of a ‘-1 triplet’ added immediately upstream of the start codon. The expression data shows that the influence of the ‘-1 triplet’ nucleotide composition varies between minimal to significant influence. Filter binding assays suggest that the influence of the ‘-1 triplet’ sequence on initial mRNA-ribosome association was minimal. Toeprint assays, that measure stable ternary complex, show a direct correlation between decreased expression and decreased ribosome binding to the 5’-recessed AUG start codon. Competition toeprint assays reveal less stability for the ‘-1 triplet’ mRNAs with low rates of ternary complex formation. Photo-crosslinking assays were used to further investigate the ribosome-5’-AUG interactions contributing to the formation of stable ternary complex. Our results suggest that E. coli ribosomes interact with a 5’-AUG at several sites and ultimately form stable ternary complexes with the 5’-AUG at ribosomal P-site.
Keywords: Leaderless mRNA, Translation initiation, mRNA ribosome interactions
